1. Field of the Invention
The present invention relates to a total internal reflection fluorescence microscope for performing fluorescence observation using evanescent illumination which is generated by total internal reflection illumination.
2. Description of the Background Art
Recently, functional analysis of biological cells has been carried out with enthusiasm. In this type of functional analysis much attention is given to the total internal reflection fluorescence microscope (TIRFM) which obtains total internal reflection images from the cell membrane and surrounding areas, as the microscope for observing cell membrane function.
When the total internal reflection fluorescence microscope totally reflects illumination light at an interface of a cover glass and a specimen, a fluorescent material is excited using light called evanescent light which permeates the limited range of several hundred nm or less at the specimen side. Thus, since only fluorescence in this limited range at the vicinity of the cover glass is observed, the background is extremely dark and high contrast fluorescence observation or weak fluorescence observation are possible.
Meanwhile, at biological research labs which use this type of total internal reflection fluorescence microscope, there are often cases where there is need to observe the in-plane which is not as deep as the interface vicinity with good contrast, as well as cases where the illumination light must reach certain depth and thus wide-range observation needs to be performed. As a result, it is preferable that the permeation depth of the evanescent light is changeable in accordance with the object for observation.
A permeation depth of evanescent light from interfaces is disclosed in Daniel Axelrod “5. Total Internal Reflection Fluorescence at Biological Surfaces”, Noninvasive Techniques in Cell Biology: 93-127, 1990, Wiley-Liss, Inc. pp. 111-113, and it is known that the following formula holds true:d=λ/4π{(n12sin θ12−n22)}1/2  (1)wherein, d is the permeation depth of evanescent light, λ is the wavelength of light, n1 is the refractive index at the incidence side (cover glass), θ1 is the incident angle, and n2 is the refractive index at the exit side (specimen).
Note that the irradiation angle for the specimen NA is obtained as follows.NA=sinθ1·n1.
As is evident from the above formula, the larger the incident angle of the illumination light for the interface of the total reflection irradiation angle, or in other words, the inclination angle of the illumination light for the vertical line of the interface, the deeper the permeation depth for the evanescent light will be.
An example of technology using this type of concept is Jpn. Pat. Appln. KOKAI Publication No. 2002-31762 in which the position of the focal point of the focal light at the rear side focal point in-plane of the objective lens is adjusted by rotation of the reflecting optical system, and the incident angle of the light introduced at the specimen side can be thereby adjusted.